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  • RESEARCH ARTICLE

    Epidermal epidemic: unravelling the pathogenesis of chytridiomycosis Nicholas C. Wu1, Rebecca L. Cramp1, Michel E. B. Ohmer2 and Craig E. Franklin1,*

    ABSTRACT Chytridiomycosis, a lethal fungal skin disease of amphibians, fatally disrupts ionic and osmotic homeostasis. Infected amphibians increase their skin shedding rate (sloughing) to slow pathogen growth, but the sloughing process also increases skin permeability. Healthy amphibians increase active ion uptake during sloughing by increasing ion transporter abundance to offset the increased skin permeability. How chytridiomycosis affects the skin function during and between sloughing events remains unknown. Here, we show that non-sloughing frogs with chytridiomycosis have impaired cutaneous sodium uptake, in part because they have fewer sodium transporters in their skin. Interestingly, sloughing was associated with a transient increase in sodium transporter activity and abundance, suggesting that the newly exposed skin layer is initially fully functional until the recolonization of the skin by the fungus again impedes cutaneous function. However, the temporary restoration of skin function during sloughing does not restore ionic homeostasis, and the underlying loss of ion uptake capacity is ultimately detrimental for amphibians with chytridiomycosis.

    KEYWORDS: Anuran, Amphibian, Batrachochytrium dendrobatidis, Electrophysiology, Epithelial disruption, Pathogen, Skin sloughing

    INTRODUCTION Amphibians are the most threatened class of vertebrates, with approximately 30% globally classified as threatened with extinction (IUCN, 2017). Although anthropogenic disturbances are the main cause of amphibian declines (Stuart et al., 2004), roughly 27% of these declines have occurred in pristine habitats such as protected national parks (Pimm et al., 2014). Many of these non- anthropogenic declines have been attributed to a cutaneous pathogen, Batrachochytrium dendrobatidis (Bd), which infects the keratinised layers of amphibian skin and can cause the disease chytridiomycosis (Berger et al., 1998; Pessier et al., 1999). Amphibian skin is unique amongst tetrapods given its high permeability, which allows it to serve a variety of physiological roles such as cutaneous gas exchange and osmotic and ionic regulation, but this necessitates a moist epidermis and so the active secretion of aqueous substances from the dermal glands renders amphibians more susceptible to relatively high rates of evaporative water loss (Boutilier et al., 1992; Larsen et al., 2014). Given the importance of physiological regulation via the skin, a disruption in

    cutaneous function tends to have serious consequences for amphibian health (Pessier, 2002).

    Amphibians with chytridiomycosis display altered behaviour (lethargy, lack of appetite) and suffer physical damage to the skin (cutaneous erythema, hyperkeratosis), leading to the loss of physiological homeostasis (low electrolyte levels) (Berger et al., 2005; Voyles et al., 2012; Peterson et al., 2013). In healthy frogs, electrolytes are exchanged through paracellular spaces or transcellularly via ion transport proteins (channels, co-transporters, exchangers, ATPase) in cutaneous epithelial cells (Hillyard et al., 2008). In frogs with chytridiomycosis, low levels of circulating electrolytes (hyponatremia and hypochloremia) correlate with a loss of cutaneous ion uptake capacity (Voyles et al., 2009). Bd produces a complex mixture of proteins (proteases, biofilm-associated proteins and a carotenoid ester lipase) that can disrupt epidermal intercellular junctions (Brutyn et al., 2012) and suppress genes related to the production of keratin and collagen (Rosenblum et al., 2012). In addition to its effects on skin integrity, Campbell et al. (2012) suggested that cutaneousBd infections may directly inhibit the epithelial sodium channels (ENaC), which are primarily responsible for the cutaneous re-uptake of Na+ from cutaneous secretions and/or from the environment (Schild, 2010; Larsen and Ramløv, 2013). This hypothesis was based on their observation that the amiloride-sensitive short-circuit current of the skin of infected frogs was lower than that of healthy frogs (Voyles et al., 2009). Dysfunction of ENaC transport is often associated with disorders of Na+ and fluid homeostasis, and blood pressure (Schild, 2004). HowBd influences cutaneous ion uptake pathways, specifically those relating to Na+ movement, including ENaC and Na+/K+-ATPase (NKA), which is responsible for generating electrochemical gradients in the epidermis (Lingrel and Kuntzweiler, 1994), remains unknown.

    Given the vital role of the skin in sustaining amphibian homeostasis, maintaining skin integrity and function is of considerable importance. To this end, the outer keratinised layer is periodically removed and replaced by ‘sloughing’. Sloughing also plays an important role in regulating cutaneous microbe abundances (Meyer et al., 2012; Cramp et al., 2014), and in frogs infected with Bd, sloughing helps remove Bd from the keratinised layer (Ohmer et al., 2017). Indeed, Ohmer et al. (2017) found that sloughing reduced Bd load in five anuran species, with less susceptible species clearing infection. However, susceptible species continued to develop chytridiomycosis despite an increase in sloughing frequency, and in spite of a temporary reduction in Bd load after sloughing (Ohmer et al., 2017). An increase in sloughing frequency might act as an immune response to remove the pathogen before the onset of disease (Ohmer et al., 2015). Importantly though, increased sloughing frequency may be a double-edged sword, as sloughing itself causes transient osmoregulatory disruption in amphibians (Jørgensen, 1949; Wu et al., 2017). In healthy amphibians (Rhinella marina), sloughing is accompanied by an increase in cutaneous permeability (Wu et al., 2017). However, an increase in the expression and activity of epithelial Na+ channels offsets theReceived 5 September 2018; Accepted 5 December 2018

    1School of Biological Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia. 2Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

    *Author for correspondence (c.franklin@uq.edu.au)

    N.C.W., 0000-0002-7130-1279; R.L.C., 0000-0001-9798-2271; M.E.B.O., 0000- 0002-5937-6585; C.E.F., 0000-0003-1315-3797

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    © 2019. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2019) 222, jeb191817. doi:10.1242/jeb.191817

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    mailto:c.franklin@uq.edu.au http://orcid.org/0000-0002-7130-1279 http://orcid.org/0000-0001-9798-2271 http://orcid.org/0000-0002-5937-6585 http://orcid.org/0000-0002-5937-6585 http://orcid.org/0000-0003-1315-3797

  • temporary increase in skin permeability such that animals suffer no loss of physiological homeostasis (Wu et al., 2017). Conversely, in green tree frogs (Litoria caerulea) with high Bd loads, cutaneous ion loss is substantially elevated during non-sloughing periods and increases further during sloughing (Wu et al., 2018). Given that animals slough more frequently, the cumulative impact of sloughing and increased ion loss during non-sloughing periods leads to the loss of physiological homeostasis. Although it is clear that Bd affects cutaneous ion transport processes in infected frogs, the mechanistic basis for the disruption of ion transport, and the effects of sloughing on this, remains unclear. Understanding how the disruption of skin function leads to the

    loss of physiological homeostasis in frogs with chytridiomycosis, and whether sloughing worsens cutaneous regulation, is important from a management standpoint. A greater understanding of the mechanistic basis for loss of homeostasis in infected frogs could lead to better treatment options, particularly for critically endangered species and captive insurance populations. Thus, the aim of this study was to investigate the effects of Bd on cutaneous ion transport processes, focusing on the abundance, activity, and expression of ENaC and NKA in a susceptible species, the Australian green tree frog. We hypothesised that both increased skin permeability and the reduction or inhibition of regulatory ion transporters resulting from Bd infection would contribute to the disruption of cutaneous ion flow, and sloughing would further exacerbate electrolyte permeability in infected frogs. The resulting disruption of both chytridiomycosis and sloughing could prolong electrolyte imbalance, causing conditions of low ion concentrations in the blood plasma such as hyponatremia.

    MATERIALS AND METHODS Animal collection and maintenance Litoria caerulea (White 1790) spawn was collected from Bribie Island, southeast Queensland, Australia, in March 2015, and raised in the laboratory at The University of Queensland until metamorphosis. The resulting 20 juveniles (10–20 g) were used for experimentation. An additional 17 L. caerulea (15–70 g) adults and subadults were collected from wet roads in non-protected areas near Fernvale, southeast Queensland, Australia, in January 2015. Frogs were housed individually in either small (235×170×120 mm) or large (265×235×12 mm) ventilated clear plastic containers, with paper towels saturated with chemically aged water (dilution 1:4000; VitaPet, NSW, Australia) as substrate, and a half PVC pipe for shelter. The lighting conditions were set at a 12 h:12 h light:dark photoperiod cycle, and temperature was set at a constant 20.5± 0.5°C. Frogs were checked daily and fed once a week on vitamin- dusted crickets (Acheta domesticus), and enclosures were cleaned weekly. Prior to experiments, all frogs were swabbed to confirm the absence of Bd infection given its widespread distribution in natural L. cae